Staphylococcus aureus leukocidins, therapeutic compositions, and uses thereof

ABSTRACT

Disclosed herein are isolated and purified  Staphylococcus aureus  bi-component leukocidin, referred to herein as LukAB, and its components LukA and LukB, antibodies specific to LukA, antibodies specific to LukB, therapeutic compositions containing LukA and/or LukB, or anti-LukA and/or anti-LukB antibodies, uses of the compositions to treat acute inflammatory conditions or  S. aureus  infection, methods for identifying inhibitors of LukAB-mediated cytotoxicity of human phagocytes, and methods for using LukAB as a marker to predict severity of  S. aureus  infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. §371 ofPCT Application No. PCT/US2011/035354, filed May 5, 2011, which claimsthe benefit of the filing date of U.S. Provisional Patent ApplicationNo. 61/331,550 filed May 5, 2010, the disclosure of which is herebyincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on May 2, 2011 is named SequenceListing_Staphylococcus Aureus Leukocidins_ST25.txt, and is 102 kilobytesin size.

BACKGROUND OF THE INVENTION

Staphylococcus aureus bacteria or “staph” are normally found on the skinor in the nose of people and animals. Staph bacteria are generallyharmless, unless they enter the body through a cut or other wound.Typically, staph infections are minor skin problems in healthy people.Historically, staph infections were treated by broad-spectrumantibiotics, such as methicillin. Now, though, certain strains of staphhave emerged that are resistant to methicillin and other β-lactamantibiotics such as penicillin and cephalosporins. They are referred toas methicillin-resistant Staphylococcus aureus (also known as multi-drugresistant Staphylococcus aureus, or “MRSA”).

Staph infections, including MRSA, generally start as small red bumpsthat resemble pimples, boils or spider bites. These bumps or blemishescan quickly turn into deep, painful abscesses that require surgicaldraining. Sometimes the bacteria remain confined to the skin. Onoccasion, they can burrow deep into the body, causing potentiallylife-threatening infections in a broad range of human tissue, includingskin, soft tissue, bones, joints, surgical wounds, the bloodstream,heart valves, lungs, or other organs. Thus, S. aureus infections canresult in potentially fatal diseases such as necrotizing fasciitis,pneumonia, endocarditis, sepsis, toxic shock syndrome, and various formsof pneumonia. MRSA infection is especially troublesome in hospital ornursing home settings where patients are prone to open wounds, invasivedevices, and weakened immune systems and thus are at greater risk forinfection than the general public. Workers who do not follow propersanitary procedures may transfer MRSA bacteria from one patient toanother.

S. aureus produces a diverse array of virulence factors and toxins thatenable this bacterium to neutralize and withstand attack by differentkinds of immune cells, specifically subpopulations of white blood cellsthat make up the body's primary defense system. The production of thesevirulence factors and toxins allow S. aureus to maintain an infectiousstate. See, Nizet, J. Allergy Clin. Immunol. 120:13-22 (2007). Amongthese virulence factors, S. aureus produces several bi-componentleukotoxins, which damage membranes of host defense cells anderythrocytes by the synergistic action of two non-associated proteins orsubunits. See, Supersac, et al., Infect. Immun. 61:580-7 (1993). Amongthese bi-component leukotoxins, gamma-hemolysin (HlgAB and HlgCB) andthe Pantone-Valentine Leukocidin (PVL) are the best characterized.

The toxicity of the leukocidins towards mammalian cells involves theaction of two components. The first subunit is named class S-subunit(i.e., “slow-eluted”), and the second subunit is named class F-subunit(i.e., “fast-eluted”). The S- and F-subunits act synergistically to formpores on white blood cells including monocytes, macrophages, dendriticcells and neutrophils (collectively known as phagocytes). See,Menestrina, et al., Toxicol. 39:1661-1672 (2001). The mechanism by whichthe bi-component toxins form pores in target cell membranes is notentirely understood. The proposed mechanism of action of these toxinsinvolves binding of the S-subunit to the target cell membrane, mostlikely through a receptor, followed by binding of the F-subunit to theS-subunit, thereby forming an oligomer which in turn forms a pre-porethat inserts into the target cell membrane (Jayasinghe, et al., Protein.Sci. 14:2550-2561 (2005)). The pores formed by the bi-componentleukotoxins are typically cation-selective. Pore formation causes celldeath via lysis, which in the cases of the target white blood cells, hasbeen reported to result from an osmotic imbalance due to the influx ofcations (Miles, et al., Biochemistry 40:8514-8522 (2001)).

In addition to PVL (also known as leukocidin S/F-PV or LukSF-PV) andgamma-hemolysin (HlgAB and HlgCB), the repertoire of bi-componentleukotoxins produced by S. aureus is known to include leukocidin E/D(LukED) and leukocidin M/F′(LukMF′). Thus, the S-class subunits of thesebi-component leukocidins include HlgA, HlgC, LukE, LukS-PV, and LukM,and the F-class subunits include HlgB, LukD, LukF-PV, and LukF′-PV(Menestrina, et al., supra.). The S. aureus S- and F-subunits are notleukocidin-specific. That is, they are interchangeable such that otherbi-component combinations could make a functional pore in a white bloodcell, greatly increasing the repertoire of leukotoxins (Meyer, et al.,Infect. Immun. 77:266-273 (2009)).

Designing effective therapy to treat MRSA infection has been especiallychallenging. In addition to the aforementioned resistance to methicillinand related antibiotics, MRSA has also been found to have significantlevels of resistance to macrolides (e.g., erythromycin), beta-lactamaseinhibitor combinations (e.g., Unasyn, Augmentin) and fluoroquinolones(e.g., ciprofloxacin), as well as to clindamycin,trimethoprim/sulfamethoxisol (Bactrim), and rifampin. In the case ofserious S. aureus infection, clinicians have resorted to intravenousvancomycin. However, there have been reports of S. aureus resistance tovancomycin. Thus, there is a need to develop new antibiotic drugs thateffectively combat S. aureus infection.

BRIEF SUMMARY OF THE INVENTION

Applicants have discovered and characterized another bi-component memberof the native Staphylococcus aureus defense system. The newlycharacterized native S-subunit polypeptide component is referred toherein as “LukA”, which embraces the native polypeptides and analogsthereof having a sequence similarity of at least 70% with the sequencesof the native polypeptides. Thus, an aspect of the present invention isdirected to an isolated and/or purified LukA. Another aspect of thepresent invention is directed to an isolated and/or purifiedpolynucleotide encoding LukA, a transformed host (e.g., cell) containingthe polynucleotide, and methods for preparation of recombinant LukA viaexpression of the polynucleotide in the transformed host.

The newly characterized F-subunit polypeptide component is referred toherein as “LukB”, which embraces the native polypeptides and analogsthereof having a sequence similarity of at least 70% with the sequencesof the native polypeptides. Thus, another aspect of the presentinvention is directed to an isolated and/or purified LukB. Anotheraspect of the present invention is directed to an isolated and/orpurified polynucleotide encoding LukB, a transformed host (e.g., cell)containing the polynucleotide, and methods for preparation ofrecombinant LukB via expression of the polynucleotide in the transformedhost.

Yet another aspect of the present application is directed to therapeuticcompositions useful in inhibiting onset of or treating a Staphyloccocusaureus infection containing therapeutically effective amounts of LukAand/or LukB formulated in a pharmaceutically acceptable carrier. Thus,in one embodiment, the therapeutic composition contains atherapeutically effective amount of LukA. In another embodiment, thetherapeutic composition contains a therapeutically effective amount ofLukB. In yet another embodiment, the therapeutic composition containstherapeutically effective amounts of both LukA and LukB. In yet otherembodiments, the composition contains an analog of LukA that lacks the10 C-terminal residues and which is non-toxic (referred to herein asLukAΔ10C or rLukAΔ10C). These compositions have multiple therapeuticuses. In some embodiments, the compositions are referred to asanti-inflammatory compositions and may be used to treat acuteinflammatory conditions or disorders, particularly localized acuteinflammatory conditions.

These uses exploit Applicants' additional discoveries that underphysiological conditions (i.e., LukAB produced directly by S. aureus),the LukAB complex has exquisite specificity for phagocytes but not othernucleated cells such as epithelial cells and endothelial cells. That is,the complex forms pores in membranes of these kinds of cells, thuscausing cell death, which is referred to herein as “LukAB-mediatedcytotoxicity.” On the other hand, LukAB has relatively little ornegligible specificity with respect to other nucleated mammalian cells.Thus, the anti-inflammatory compositions of the present inventionexploit the specificity of LukAB for human phagocytes, for purposes oftreating acute inflammatory conditions, which are characterized bymassive infiltration of phagocytes to the site of inflammation.

In other embodiments, the therapeutic compositions may be referred to asa (active) vaccine composition. The compositions may be used to induceproduction of neutralizing anti-LukA and anti-LukB antibodies in asubject at risk of S. aureus infection or a subject diagnosed with S.aureus infection such as MRSA.

Other aspects of the present invention are directed to antibodies thatspecifically bind LukA, antibodies that specifically bind LukB,therapeutic compositions containing the LukA and/or LukB antibodies, anduses thereof to treat S. aureus infectious conditions. These therapeuticcompositions may be referred to as passive vaccine compositions. Thus,in one embodiment, the therapeutic composition contains atherapeutically effective amount of anti-LukA antibodies. In anotherembodiment, the therapeutic composition contains a therapeuticallyeffective amount of anti-LukB antibodies. In yet another embodiment, thetherapeutic composition contains therapeutically effective amounts ofboth anti-LukA and anti-LukB antibodies.

The passive and active vaccine compositions of the present inventionexploit Applicants' further discovery that infectious, virulent S.aureus strains such as MRSA, express LukA and LukB. The conservation ofLukA and LukB across a large spectrum of S. aureus strains enables thevaccines of the present invention to provide full-spectrum therapeuticeffectiveness. LukA, LukB, anti-LukA antibodies and anti-LukB antibodiesare also referred to herein as active agents.

A further aspect of the present invention is directed to methods ofusing LukAB, LukA, and/or LukB to identify potential inhibitors ofLukAB-mediated cytotoxicity. These methods may utilize the LukABcomplex, per se, in combination with a phagocyte, or a phagocytemembrane-binding portion thereof. Thus-identified inhibitors may becandidates for therapy for the purposes of treating S. aureus infection.

An even further aspect of the present invention is directed to a methodof predicting or assessing severity of an S. aureus infection whichentails detecting presence or amount of LukA and/or LukB, or detectingcorresponding genes of LukA and/or LukB, in a biological sample obtainedfrom an infected subject. This aspect of the present invention is basedon Applicants' even further discovery that among the many cytotoxinsproduced by S. aureus, LukAB exhibits potent toxicity towards humanphagocytes. Thus, detection of presence or relatively high amounts ofLukA and/or LukB, or their corresponding genes (e.g., as exhibited by S.aureus strain Newman, 4645, and MRSA strains USA300 and USA500) relativeto a control (e.g., S. aureus strains USA100 and USA400) which produceslittle or undetectable amounts of LukA and/or LukB, is indicative of asevere S. aureus infection.

These and other aspects of the present invention are more fullydescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment containing the amino acid sequence of a majorityLukA sequence (designated as SEQ ID NO:1), and the LukA polypeptides,LukA (Newman).pro (SEQ ID NO: 2), HMPREF0772_0044(THC60).pro (SEQ ID NO:3), HMPREF0774_2356(TCH130) .pro (SEQ ID NO: 4),HMPREF0776_0173(USA300_TCH959).pro (SEQ ID NO: 5), MW1942(MW2).pro (SEQID NO: 6), SA1813 (N315).pro (SEQ ID NO: 7), SAB1876c (RF122).pro SEQ IDNO: 8), SACOL2006 (Col).pro (SEQ ID NO: 9), SALG_02329 (A9635).pro (SEQID NO: 10), SAPIG2061 (ST398).pro (SEQ ID NO: 11), SAR2108 (MRSA252).pro(SEQ ID NO: 12), SATG_01930 (D139).pro (SEQ ID NO: 13), and SAV2005(Mu50).pro (SEQ ID NO: 14), from thirteen (13) different strains of S.aureus to which it corresponds.

FIG. 2 is an alignment containing the amino acid sequence of a majorityLukB sequence (designated as SEQ ID NO:15), and the LukB polypeptides,A9635.pro (SEQ ID NO: 16), COL.pro (SEQ ID NO: 17), D139.pro (SEQ ID NO:18), E1410.pro (SEQ ID NO: 19), JKD6008.pro (SEQ ID NO: 20), MRSA252.pro(SEQ ID NO: 21), Mu50.pro (SEQ ID NO: 22), MW2.pro (SEQ ID NO: 23),RF122.pro (SEQ ID NO: 24), TCH130.pro (SEQ ID NO: 25),USA300_FPR3757.pro (SEQ ID NO: 26), and Newman (LukB).pro (SEQ ID NO:27), from twelve (12) different strains of S. aureus to which itcorresponds.

FIG. 3. LukAB is a potent staphylococcal cytotoxin that targets andkills primary human phagocytes. (a) Intoxication of primary humanperipheral blood mononuclear cells (PBMCs) with culture filtrate (2.5%v/v) from S. aureus strain Newman (WT) and the indicated isogenic mutantstrains. Cell viability was monitored using a commercially availablecell viability assay, where cells treated with medium were set at 100%.Results represent the average of triplicate samples+standard deviation(S.D.). (b) Intoxication of primary human monocytes, macrophages, anddendritic cells (DC) with culture filtrate (2.5% v/v) from S. aureusstrain Newman (WT) and the indicated isogenic mutant strains. Cellviability was monitored as described above. Results represent the meanfrom two donors, where cells from each donor were intoxicated with threeindependent exoprotein preparations, +S.E.M. (c) Intoxication of primaryhuman PMNs with various dilutions of culture filtrates from the S.aureus strain Newman (WT) and the indicated isogenic mutant strains.Cell viability was monitored as described above. Results represent themean from PMNs isolated from four donors ±S.E.M. (d) Intoxication ofprimary human PMNs with purified rLukA, rLukB, or a combination of rLukAand rLukB at the indicated concentrations. * indicates statisticalsignificance from both rLukA and rLukB, P<0.05. For panels (a-c) *indicates statistical significance from WT, ** indicates statisticalsignificance from ALukAB/p, P<0.05(Student's t test p<0.05).

FIG. 4. LukAB preferentially targets human phagocytic cells.Intoxication of (a, c and d) PMN-HL60 or (b) THP1 cells with variousdilutions of culture filtrate from the S. aureus WT strain Newman,isogenic mutant strains lacking the indicated genes/toxins, (c) culturefiltrate from S. aureus WT containing an empty plasmid (WT/p),a strainlacking LukAB with and empty plasmid (ΔLukAB/p), and a strain lackingLukAB with a LukAB complementation plasmid(ΔLukAB/pLukAB), or (d) withpurified recombinant LukA(rLukA), LukB (rLukB), or a combination ofrLukA and rLukB (rLukA+rLukB) at the indicated concentrations. For theintoxications with both rLukA and rLukB, the total protein concentrationis comprised of equal amounts of rLukA and rLukB(e.g. 2.8 μg totalprotein is equal to 1.4 μg of rLukA and 1.4 μg of rLukB). (e)Intoxication of the indicated human cell lines with 10 μg/m1 of rLukAB.Cell viability was monitored using a commercially available cellviability assay, where cells treated with medium were set at 100%.Results represent the average of triplicate samples ±S.D. Asterisk (*)denote statistically significant difference compared to WT (One-wayANOVA).

FIG. 5. LukAB is an important toxin in different staphylococcal strains.(A) Expression of LukB by various S. aureus strains as determined byWestern blot analysis using an anti-LukB polyclonal sera. (B)Intoxication of PMN-HL60 with dilutions of exoproteins from different S.aureus strains. Cell viability was monitored using a commerciallyavailable cell viability assay, where cells treated with medium were setat 100% viable. (C) Expression of LukB and α-toxin by WT and LukABisogenic strains as determined by Western blot analysis usingtoxin-specific sera. (D) Intoxication of PMN-HL60 with exoproteins fromWT strains Newman (New.) and 4645, and the LukAB isogenic strains. Cellviability was monitored as in Panel B. Results represent the average oftriplicate samples +S.D. * denote statistically significant differencecompared to Newman (C) or to WT (E) (Student's t test p<0.05).

FIG. 6. LukAB disrupts the plasma membranes of target cells. (a) Lightmicroscopy images of PMN-HL60 cells intoxicated with culture filtratefrom the S. aureus WT strain and the isogenic strain lacking LukAB(ΔLukAB). (b-c) Intoxication of PMN-HL60 cells with culture filtratesfrom the WT strain (WT/p), the isogenic strain lacking LukAB (ΔLukAB/p),the complemented strain (ΔLukAB/pLukAB), or mock intoxicated withmedium. Cells with compromised membranes were stained with SYTOX Green,imaged by fluorescence microscopy (c), and green-fluorescence intensitywas measured (b). (d) PMNs were infected ex vivo with S. aureus strainNewman (MSSA) or USA300 strain LAC (MRSA) and the indicated isogenicmutants at various multiplicities of infection (MOI). Membrane damagewas monitored with SYTOX green. Results represent the average oftriplicate samples±S.D. Asterisks (*) denote statistically significantdifference compared to WT (Student's t test p<0.05).

FIG. 7. LukAB protects S. aureus from host-mediated killing by targetingand killing phagocytes. (a) Infection of PMN-HL60 cells with S. aureusWT, a strain lacking lukAB, and the strain lacking lukAB with a lukABcomplementation plasmid (ΔlukAB/plukAB) at various multiplicities ofinfection (MOI). Mammalian cells with compromised membranes weremonitored with SYTOX Green as described in FIG. 6. Results represent theaverage of triplicate samples+S.D. (b) Viability of the indicated S.aureus strains upon ex vivo infection of human whole blood. Resultsrepresent the mean from whole blood isolated from 12 donors+S.E.M. (c)Viability of the indicated S. aureus Newman strains upon infection ofprimary human neutrophils (PMNs). Results represent the mean from PMNsisolated from 12 donors+S.E.M. (d) Intoxication of primary human PMNswith various dilutions of culture filtrate from the WT/p, ΔlukAB/p, andΔlukAB/plukAB strains. LDH-release was measured as an indicator of celllysis. Results represent the mean from PMNs isolated from 6donors+S.E.M. Asterisks (*) denote statistically significant differencecompared to WT strain Newman (Student's t test p<0.05).

FIG. 8. LukAB is important for the pathogenesis of S. aureus in vivo.(a) Bioluminescent images of kidneys from mice infected with the WT S.aureus strain LAC containing pXen1 or the pLukAB.Xen1. The kidneys oftwo representative mice per group are shown. (b) Bacterial loadrecovered from the kidneys of mice infected retro-orbitally with theindicated S. aureus LAC strains. Each data point represents the numberof bacteria (CFU) per milliliter of tissue homogenate in a singleanimal. Dashed line indicates the limit of detection. For panels (A-Cand E) * indicates statistical significance from WT, ** indicatesstatistical significance from ΔLukAB/p, P<0.05.

FIG. 9. LukAB kills human phagocytes by forming pores on cell membranes.(a) PMN-HL60 cells were intoxicated with rLukA+rLukB and toxin bindingwas monitored by SDS-PAGE and immunoblotting using antibodies specificfor LukA or LukB. (b) PMN-HL60were incubated with rLukAB and toxinbinding determined by FACS using a rabbit anti-His antibody. (c)PMN-HL60 cells were intoxicated with rLukAB and the formation of LukABoligomers in the plasma membrane was determined by SDS-PAGE andimmunoblotting using an anti-LukB antibody. (d) PMN-HL60 intoxicatedwith rLukAB or treated with saponin in the presence or absence ofPEG-400. LukAB pores were detected with ethidium bromide. (e) Viabilityof PMN-HL60 treated as in panel determined with a commercially availablecell viability assay, where cells treated with medium were set at 100%.Results in panels d and e represent the average of triplicate samples +/SEM. * denote statistically significant difference to —PEGs (panel d-e)(Student's t test p<0.05).

FIG. 10. LukAB cytotoxicity can be neutralized by an α-LukA polyclonalantibody. Intoxication of PMN-HL60s with 5% (v/v) culture filtrate fromS. aureus strain Newman that had been incubated with the indicatedamounts of α-LukA polyclonal antibodies or pre-immune serum from variousproduction bleeds from two different rabbits. Cell viability wasmonitored using a commercially available cell viability assay, wherecells treated with medium were set at 100%. Results represent theaverage of triplicate samples ±standard deviation (S.D.).

FIG. 11. The LukA C-terminal extension is necessary for the cytotoxiceffect of LukAB but is not needed for recognition by an α-LukApolyclonal antibody. (a) Alignment of amino acid sequences from thevarious S. aureus leukotoxin S-subunits (designated as SEQ ID NOS:44-49)performed using the MegAlign Clustal W method from Lasergene software.The N- and C-terminal extensions only present in the LukA sequence areemphasized with boxes. (b) Coomassie blue staining of 2 μg ofrecombinant LukA (rLukA), LukB (rLukB), LukA lacking the C-terminalextension (rLukAΔ10C) and LukA lacking the N-terminal extension(rΔ33NLukA) purified from E. coli and separated by SDS-PAGE accompaniedby intoxication of PMN-HL60s with various amounts of rLukA, rLukAΔ10Cand rΔ33NLukA paired with rLukB. The final protein concentrationrepresents equal amounts of rLukA, rLukAΔ10C or rΔ33NLukA and rLukB.Results represent the average of triplicate samples±S.D. (c) Immunoblotshowing equivalent recognition of 6×His-tagged rLukAΔ10C by both α-LukAand α-His polyclonal antibodies.

DETAILED DESCRIPTION

The following disclosure is directed, in successive order, to LukApolypeptides, LukB polypeptides, LukA and LukB polynucleotides,anti-LukA and anti-LukB antibodies, therapeutic compositions containingLukA and/or LukB, or anti-LukA and/or anti-LukB antibodies, methods ofusing the therapeutic compositions, methods of identifying inhibitors ofLukAB-mediated cytotoxicity, and methods of predicting or assessingseverity of an S. aureus infection.

LukA Polypeptides

Polypeptides native to Staphylococcus aureus have now been isolated andidentified by Applicants as exhibiting the activity profile of knownS-subunit leukocidins (e.g., LukS-PVL, LukE and HlgC). Thesepolypeptides which are designated collectively herein as LukA,specifically target and bind human phagocytes (but not human epithelialor human endothelial cells, or murine cells), and once bound to thephagocyte membrane, LukA oligomerizes with an S. aureus F-subunitleukocidin (e.g., LukF-PVL, LukD and HlgB, and LukB as disclosedherein), and upon oligomerization forms a transmembrane pore(collectively referred to as LukA activity). The alignment illustratedin FIG. 1 contains amino acid sequences of a majority LukA sequence(designated herein as SEQ ID NO:1), and the LukA polypeptides from 13different strains of S. aureus to which it corresponds (designatedherein as SEQ ID NOS:2-14).

The N-terminal 27 amino acid residues in each of SEQ ID NOS:1-14represent the native secretion/signal sequence. Thus, the mature,secreted form of LukA, which is represented by amino acid residues28-351 in each of SEQ ID NOS: 1-14, may be referred to herein as “LukA(28-351)” or “mature LukA”. Correspondingly, the immature form of LukAmay be referred to herein as “LukA (1-351)”.

A LukA consensus sequence, based on SEQ ID NOS: 2-14 (which are notexhaustive with respect to native S. aureus LukA) would thus includevariability at a minimum of 64 positions of LukA (wherein consecutivepositions of variability are denoted X¹-X⁶⁴, designated as follows:8(X¹=L or F), 16 (X²=A or V), 17 (X³=I or L), 24 (X⁴=T or N), 26 (X⁵=Qor E), 31 (X⁶=H or N), 38 (X⁷=N or T), 46 (X⁸=S or A), 50 (X⁹=E or D),55 (X¹⁰=T or N), 56 (X¹¹=N or D), 61 (X¹²=S or T), 62 (X¹³=T or P), 63(X¹⁴=A, G or V), 73 (X¹⁵=I or V), 78 (X¹⁶=E or V), 77 (X¹⁷=T or S), 80(X¹⁸=V or E), 83 (X¹⁹=E or K), 84 (X²⁰=E or K), 105 (X²¹=V or I), 124(X²²=K or R), 125 (X²³=E or N), 127 (X²⁴=K, T or N), 129 (X²⁵=S or A),130 (X²⁶=N or S), 135 (X²⁷=K or Q) 146 (X²⁸=R or S), 148 (X²⁹=R or P),173 (X³⁰=S or N), 174 (X³¹=S or L), 181 (X³²=T or V), 184 (X³³=I or V),195 (X³⁴=T or S), 202 (X³⁵=N or K), 208 (X³⁶=S or I), 214 (X³⁷=W or R),221 (X³⁸=I or V) 229 (X³⁹=G or N), 231 (X⁴⁰=V or I), 237 (X⁴¹=E or D),239 (X⁴²=L or F), 243 (X⁴³=N or T), 246 (X⁴⁴=I or L), 247 (X⁴⁵=A or 8),278 (X⁴⁶=L or I), 283 (X⁴⁷=S or T), 285 (X⁴⁸=E or D), 288 (X⁴⁹=Q or R)299 (X⁵⁰=I or V), 303 (X⁵¹=R or K), 309 (X⁵²=A or G), 310 (X⁵³=P or Q),315 (X⁵⁴=K or Q), 318 (X⁵⁵=D or E), 322 (X⁵⁶=L or F), 325 (X⁵⁷=T or V),338 (X⁵⁸=V or I), 339 (X⁵⁹=D or E), 342 (X⁶⁰=S or T), 344 (X⁶¹=D, E orQ), 347 (X⁶²=P or S), 348 (X⁶³=Y or F), and 349 (X⁶⁴=K or R).

LukB Polypeptides

Polypeptides native to Staphylococcus aureus have now been identified byApplicants as exhibiting the activity profile of known F-subunitleukocidins (e.g., LukF-PVL, LukD and HlgB). These polypeptides whichare designated collectively herein as LukB, specifically oligomerizewith an S. aureus S-subunit leukocidin (e.g., LukS-PVL, LukE and HlgC,and LukA as disclosed herein) which is bound to a human phagocyte; andupon oligomerization form a transmembrane pore in the phagocyte(collectively referred to as LukB activity). The alignment illustratedin FIG. 2 contains amino acid sequences of a majority LukB sequence(designated herein as SEQ ID NO:15) and the LukB polypeptides from the12 different strains of S. aureus to which it corresponds (designatedherein as SEQ ID NOS:16-27).

The N-terminal 29 amino acid residues in each of SEQ ID NOS:15-27represent the secretion/signal sequence. Thus, the mature, secreted formof LukB, which is represented by amino acid residues 30-339 in each ofSEQ ID NOS: 16-27, may be referred to herein as “LukB (30-339)” or“mature LukB”. Correspondingly, the immature form of LukB may bereferred to herein as “LukA (1-339)”.

A LukB consensus sequence, based on SEQ ID NOS:15-28 (which are notexhaustive with respect to native S. aureus LukB) would thus includevariability at a minimum of 49 positions of LukB (wherein consecutivepositions of variability are denoted X¹-X⁴⁹), designated as follows: 5(X¹=L or V), 6 (X²=C or Y), 13 (X³=S or T), 15 (X⁴=A or T), 16 (X⁵=L orI), 19 (X⁶=A or T), 20 (X⁷=L or F), 23 (X⁸=F or L), 26 (X⁹=S or T), 28(X¹⁰=Y or F), 34 (X¹¹=E or K), 36 (X¹²=K or T), 37 (X¹³=Q, T or A), 46(X¹⁴=D or E), 59 (X¹⁵=S or T), 60 (X¹⁶=Q or E), 62 (X¹⁷=N or K), 64(X¹⁸=T or S), 75 (X¹⁹=P or K), 95 (X²⁰=K or R), 98 (X²¹=N, d or E), 126(X²²=S or a deletion), 159 (X²³=R or Q), 163 (X²⁴=T or P), 170 (X²⁵=S orK), 187 (X²⁶=L or I), 190 (X²⁷=S or P), 192 (X²⁸=S or T), 193 (X²⁹=S orT), 193 (X²⁹=H or N), 197 (X³⁰=G or A), 204 (X³¹=S or L), 222 (X³²=D orN), 224 (X³³=T or V), 247 (X³⁴=N or D), 270 (X³⁵=N or K), 272 (X³⁶=K orE), 276 (X³⁷=R, Q or K), 287 (X³⁸=D or E), 290 (X³⁹=L or I), 294 (X⁴⁰=Kor R), 309 (X⁴¹=Q or KO, 327 (X⁴²=D or N), 329 (X⁴³=L or F), 330 (X⁴⁴=Ior V), 332 (X⁴⁵=t or V), 333 (X⁴⁶=f, I or L), 336 (X⁴⁷=K or N), and 338(X⁴⁸=K or Q).

LukA and LukB leukocidins may differ from the native polypeptidesdesignated as SEQ ID NOS:2-14 and 16-27 respectively, in terms of one ormore additional amino acid insertions, substitutions or deletions, e.g.,one or more amino acid residues within SEQ ID NOS:2-14 or 16-27 may besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. That is to say,the change relative to the native sequence would not appreciablydiminish the basic properties of native LukA and LukB. Examples includeSEQ ID NOS:1 and 15. Any such analog of LukA or LukB may be screened inaccordance with the protocols disclosed herein (e.g., the cell toxicityassay and the membrane damage assay) to determine if it maintains nativeLukA or LukB activity. Substitutions within these leukocidins may beselected from other members of the class to which the amino acidbelongs. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. Polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. Positivelycharged (basic) amino acids include arginine, lysine and histidine.Negatively charged (acidic) amino acids include aspartic acid andglutamic acid.

In other embodiments, non-conservative alterations (e.g., one or aminoacid substitutions, deletions and/or additions) can be made for purposesof inactivating or detoxifying LukA and LukB. In one embodiment, thenontoxic LukA analog differs from the native polypeptides in that theC-terminal amino acids in positions 342-351 are deleted. With theexception of SEQ ID NOs:4-6 (which contain 9 amino acids at thesepositions), the analog lacks the 10 C-terminal amino acid residues.Collectively, these analogs are referred to as LukAΔ10C. The detoxifiedLukA and LukB may be used in the active vaccine compositions describedherein. Molecular alterations can be accomplished by methods well knownin the art, including primer extension on a plasmid template usingsingle stranded templates (Kunkel, Proc. Acad. Sci., USA 82:488-492(1985)), double stranded DNA templates (Papworth, et al., Strategies9(3):3-4 (1996)), and by PCR cloning (Braman, J. (ed.), IN VITROMUTAGENESIS PROTOCOLS, 2nd ed. Humana Press, Totowa, N.J. (2002).Methods of determining whether a given molecular alteration in LukA orLukB reduces LukAB cytotoxicity are described herein.

Therefore, in view of the foregoing and for purposes of the presentinvention, LukA may be more broadly described in terms of any of SEQ IDNOS:1-14 (e.g., SEQ ID NO:2, which is the LukA polypeptide native to theNewman strain of S. aureus), or a (native or non-native) polypeptidehaving at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence similarity thereto.

Likewise, in view of the foregoing and for purposes of the presentinvention, LukB may be more broadly described in terms of any of SEQ IDNOS:15-27 (e.g., SEQ ID NO:27, which is the LukB polypeptide native tothe Newman strain of S. aureus), or a (native or non-native) polypeptidehaving at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence similarity thereto.

Thus, unless indicated to the contrary, both the immature and the matureforms of native LukA and LukB, and the sequences having less than 100%similarity with native LukA (i.e., native sequences and analogs alike,collectively referred to herein as “LukA” and “LukB”) may be used in thecompositions and methods, and for making the anti-LukA and the anti-LukBantibodies of the present invention.

Polynucleotides Encoding LukA and LukA and Methods of Synthesizing orIsolating LukA and LukB

The LukA and LukB leukocidins may be synthesized via recombinant DNAmethodologies which are well known in the art. For example, a nucleotidesequence (designated SEQ ID NO:28) encoding LukA polypeptide of S.aureus (Newman) (SEQ ID NO:2), is set forth below. Degenerate sequences(e.g., that may be useful in view of codon preferences in hosts ofchoice for purposes of recombinant expression) that encode thispolypeptide, and polynucleotides that encode other LukA polypeptides areknown in the art, or may be designed by persons of skill in the art.

atgaaaaataaaaaacgtgttttaatagcgtcatcattatcattttcaattttattgtta M  K  N  K  K  R  V  L  I  A  S  S  L  S  C  A  I  L  L  Ltcagcagcaacgactcaagcaaattcagctcataaagactctcaagaccaaaataagaaa S  A  A  T  T  Q  A  N  S  A  H  K  D  S  Q  D  Q  N  K  K gaacatgttgataagtctcaacaaaaagacaaacgtaatgttactaataaagataaaaat E  H  V  D  K  S  Q  Q  K  D  K  R  N  V  T  N  K  D  K  Ntcaacagcaccggatgatattgggaaaaacggtaaaatcacaaaacgaactgaaacagta S  T  A  P  D  D  I  G  K  N  G  K  I  T  K  R  T  E  T  Vtatgatgagaaaacaaatatactccaaaatttacaattcgactttatcgatgatccaact Y  D  E  K  T  N  I  L  Q  N  L  Q  F  D  F  I  D  D  P  Ttatgacaagaatgtattacttgttaaaaaacaaggctcaattcattcaaatttaaagttt Y  D  K  N  V  L  L  V  K  K  Q  G  S  I  H  S  N  L  K  Fgaatctcataaagaagaaaaaaattcaaattggttaaagtatccaagtgagtaccatgta E  S  H  K  E  E  K  N  S  N  W  L  K  Y  P  S  E  Y  H  Vgattttcaagtaaaaagaaatcgtaaaactgaaatattagaccaattgccgaaaaataaa D  F  Q  V  K  R  N  R  K  T  E  I  L  D  Q  L  P  K  N  Katttcaactgcaaaagtagacagtacattttcatatagctcaggtggtaaattcgattca I  S  T  A  K  V  D  S  T  F  S  Y  S  S  G  G  K  F  D  Sacaaaaggtattggacgaacttcatcaaatagctactccaaaacgattagttataatcag T  K  G  I  G  R  T  S  S  N  S  Y  S  K  T  I  S  Y  N  Qcaaaattatgacacaattgccagcggtaaaaataataactggcatgtacactggtcagtt Q  N  Y  D  T  I  A  S  G  K  N  N  N  N  H  V  H  W  S  Vattgcgaatgacttgaagtatggtggagaagtgaaaaatagaaatgatgaattattattc I  A  N  D  L  K  Y  G  G  E  V  K  N  R  N  D  E  L  L  Ftatagaaatacgagaattgctactgtagaaaaccctgaactaagctttgcttcaaaatat Y  R  N  T  R  I  A  T  V  E  N  P  E  L  S  F  A  S  K  Yagatacccagcattagtaagaagtggctttaatccagaatttttaacttatttatctaat R  Y  P  A  L  V  R  S  G  F  N  P  E  F  L  T  Y  L  S  Ngaaaagtcaaatgagaaaacgcaatttgaagtaacatacacacgaaatcaagatattttg E  K  S  N  E  K  T  Q  F  E  V  T  Y  T  R  N  Q  D  I  Laaaaacagacctggaatacattatgcacctccaattttagaaaaaaataaagatggtcaa K  N  R  P  G  I  H  Y  A  P  P  I  L  E  K  N  K  D  G  Qagattaattgtcacttatgaagttgattggaaaaataaaacagttaaagtcgttgataaa R  L  I  V  T  Y  E  V  D  W  K  N  K  T  V  K  V  V  D  Ktattctgatgacaataaaccttataaagaaggataa  Y  S  D  D  N  K  P  Y  K  E  G

A nucleotide sequence (designated herein as SEQ ID NO:29) that encodesLukB polypeptide of S. aureus (Newman), (SEQ ID NO:27), is set forthbelow. Degenerate sequences (e.g., that may be useful in view of codonpreferences in hosts of choice for purposes of recombinant expression)that encode this polypeptide, and polynucleotides that encode other LukBpolypeptides are known in the art, or may be designed by persons ofskill in the art.

atgattaaacaactatgtaaaaatatcacaatttgtacgttagcactatcgactactttc M  I  K  Q  L  C  K  N  I  T  I  C  T  L  A  L  S  T  T  Factgtattaccagctacttcatttgcaaagattaattctgaaatcaaacaagtttctgag T  V  L  P  A  T  S  F  A  K  I  N  S  E  I  K  Q  V  S  Eaagaatcttgatggtgatactaaaatgtatacacgtacagctacaacaagtgatagtcaa K  N  L  D  G  D  T  K  M  Y  T  R  T  A  T  T  S  D  S  Qaaaaatattactcaaagcttacaatttaatttcttaactgaacctaattatgataaagaa K  N  I  T  Q  S  L  Q  F  N  F  L  T  E  P  N  Y  D  K  Eacagtatttattaaagcaaaaggtacaattggtagtggtttgagaattttagacccaaat T  V  F  I  K  A  K  G  T  I  G  S  G  L  R  I  L  D  P  Nggttattggaatagtacattaagatggcctggatcttattcagtttcaattcaaaatgtt G  Y  W  N  S  T  L  R  W  P  G  S  Y  S  V  S  I  Q  N  Vgatgacaacaacaatacaaatgtgactgactttgcaccaaaaaatcaggatgaatcaaga D  D  N  N  N  T  N  V  T  D  F  A  P  K  N  Q  D  E  S  Rgaagttaaatatacgtatggttataaaacaggtggagatttttcgattaatcgtggaggc E  V  K  Y  T  Y  G  Y  K  T  G  G  D  F  S  I  N  R  G  Gttaactggaaatattacaaaagagagtaattattcagagacgattagttatcaacaacca L  T  G  N  I  T  K  E  S  N  Y  S  E  T  I  S  Y  Q  Q  Ptcatatcgtacattacttgatcaatctacgtcacataaaggtgtaggttggaaagtagaa S  Y  R  T  L  L  D  Q  S  T  S  H  K  G  V  G  W  K  V  Egcacatttgataaataatatgggacatgaccatacgagacaattaactaatgatagtgat A  H  L  I  N  N  M  G  H  D  H  T  R  Q  L  T  N  D  S  Daatagaactaaaagtgaaattttttctttaacacgaaatggaaatttatgggcgaaagat N  R  T  K  S  E  I  F  S  L  T  R  N  G  N  L  W  A  K  Daatttcacacctaaagacaaaatgcctgtaactgtgtctgaagggtttaatccagaattt N  F  T  P  K  D  K  M  P  V  T  V  S  E  G  F  N  P  E  Fttagctgttatgtcacatgataaaaaagacaaaggtaaatcacaatttgttgttcattat L  A  V  M  S  H  D  K  K  D  K  G  K  S  Q  F  V  V  H  Yaaaagatcaatggatgagtttaaaatagattggaatcgccatggtttctggggctattgg K  R  S  M  D  E  F  K  I  D  W  N  R  H  G  F  W  G  Y  Wtctggtgaaaaccatgtagataaaaaagaagaaaaattataagcattatatgaagttgat S  G  E  N  H  V  D  K  K  E  E  K  L  S  A  L  Y  E  V  Dtggaagacacataatgtgaagtttgtaaaagtacttaatgataatgaaaagaaataa W  K  T  H  N  V  K  F  V  K  V  L  N  D  N  E  K  K  -

The LukA- and LukB-encoding polynucleotides may be expressed in a hostsuch as bacteria (E. coli), plants and or yeast and then isolated andpurified. Alternatively, LukA and LukB leukocidins may be isolated fromS. aureus bacteria (e.g., the Newman strain) in accordance with standardtechniques. Thus, these leukocidins may be isolated (from a native ornon-native environment). They may also be purified in that they aresubstantially free from other proteins and cell components with which S.aureus LukA and LukB are associated in their native state (i.e.,proteins and cell components present in S. aureus cells) or a non-nativestate (i.e., proteins and cell components of a recombinant cellularhost). Suitable purification schemes, which typically entail acombination of at least two successive procedures, are known in the art.See, Deutscher, Methods in Enzymology, 182 (1990); and Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, N.Y. (1982),using one or a combination of two or more standard techniques such asaffinity column chromatography and cation-exchange liquidchromatography.

Anti-LukA Antibodies and Anti-LukB Antibodies

Aspects of the present invention are directed to anti-LukA antibodiesthat specifically bind LukA, and anti-LukB antibodies that specificallybind LukB, therapeutic compositions containing the antibodies, andmethods of use thereof. For purposes of the present invention, the term“antibody” includes monoclonal antibodies, polyclonal antibodies,antibody fragments, and genetically engineered forms of the antibodies,and combinations thereof. More specifically, the term “antibody”, whichis used interchangeably with the term “immunoglobulin”, includesfull-length (i.e., naturally occurring or formed by normalimmunoglobulin gene fragment recombinatorial processes) immunoglobulinmolecules (e.g., an IgG antibody) and immunologically active fragmentsthereof (i.e., including the specific binding portion of the full-lengthimmunoglobulin molecule), which again may be naturally occurring orsynthetic in nature. Accordingly, the term “antibody fragment” includesa portion of an antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFvand the like. Regardless of structure, an antibody fragment binds withthe same antigen that is recognized by the full-length antibody, and, inthe context of the present invention, specifically binds LukA, LukB or aLukAB complex. Methods of making and screening antibody fragments arewell-known in the art.

In some embodiments, the anti-LukA antibodies of the present inventionmay have some degree of cross-reactivity with other Staphylococcusleukocidin S-subunits such as HlgC, LukS-PVL, HlgA, LukS-I, LukE, LukEv,and LukM. Likewise, in some embodiments, the anti-LukB antibodies of thepresent invention may have some degree of cross-reactivity with otherStaphylococcus leukocidin F-subunits such as LukF′-PV, LukF-PV, LukDv,LukD, LukF-I, and HlgB. Anti-LukA and/or anti-LukB antibodies mayinhibit or reduce LukA activity and LukB activity, respectively. In someembodiments, the anti-LukA and/or anti-LukB antibodies neutralize (e.g.,substantially eliminate) LukA and LukB activity, respectively.

Naturally occurring antibodies typically have two identical heavy chainsand two identical light chains, with each light chain covalently linkedto a heavy chain by an inter-chain disulfide bond and multiple disulfidebonds further link the two heavy chains to one another. Individualchains can fold into domains having similar sizes (110-125 amino acids)and structures, but different functions. The light chain can compriseone variable domain (VL) and/or one constant domain (CL). The heavychain can also comprise one variable domain (VH) and/or, depending onthe class or isotype of antibody, three or four constant domains (CHI,CH 2, CH3 and CH4). In humans, the isotypes are IgA, IgD, IgE, IgG, andIgM, with IgA and IgG further subdivided into subclasses or subtypes(IgA1-2 and IgG1-4).

Generally, the variable domains show considerable amino acid sequencevariability from one antibody to the next, particularly at the locationof the antigen-binding site. Three regions, called hyper-variable orcomplementarity-determining regions (CDRs), are found in each of VL andVH, which are supported by less variable regions called frameworkvariable regions. The inventive antibodies include IgG monoclonalantibodies but the antibodies of the present invention also includeantibody fragments or engineered forms. These are, for example, Fvfragments, or proteins wherein the CDRs and/or variable domains of theexemplified antibodies are engineered as single-chain antigen-bindingproteins.

The portion of an antibody consisting of the VL and VH domains isdesignated as an Fv (Fragment variable) and constitutes theantigen-binding site. A single chain Fv (scFv or SCA) is an antibodyfragment containing a VL domain and a VH domain on one polypeptidechain, wherein the N terminus of one domain and the C terminus of theother domain are joined by a flexible linker. The peptide linkers usedto produce the single chain antibodies are typically flexible peptides,selected to assure that the proper three-dimensional folding of the VLand VH domains occurs. The linker is generally 10 to 50 amino acidresidues, and in some cases is shorter, e.g., about 10 to 30 amino acidresidues, or 12 to 30 amino acid residues, or even 15 to 25 amino acidresidues. An example of such linker peptides includes repeats of fourglycine residues followed by a serine residue.

Single chain antibodies lack some or all of the constant domains of thewhole antibodies from which they are derived. Therefore, they canovercome some of the problems associated with the use of wholeantibodies. For example, single-chain antibodies tend to be free ofcertain undesired interactions between heavy-chain constant regions andother biological molecules. Additionally, single-chain antibodies areconsiderably smaller than whole antibodies and can have greaterpermeability than whole antibodies, allowing single-chain antibodies tolocalize and bind to target antigen-binding sites more efficiently.Furthermore, the relatively small size of single-chain antibodies makesthem less likely to provoke an unwanted immune response in a recipientthan whole antibodies.

Fab (Fragment, antigen binding) refers to the fragments of the antibodyconsisting of the VL, CL, VH, and CH1 domains. Those generated followingpapain digestion simply are referred to as Fab and do not retain theheavy chain hinge region. Following pepsin digestion, various Fabsretaining the heavy chain hinge are generated. Those fragments with theinterchain disulfide bonds intact are referred to as F(ab′)2, while asingle Fab′ results when the disulfide bonds are not retained. F(ab′)₂fragments have higher avidity for antigen that the monovalent Fabfragments.

Fc (Fragment crystallization) is the designation for the portion orfragment of an antibody that comprises paired heavy chain constantdomains. In an IgG antibody, for example, the Fc comprises CH2 and CH3domains. The Fc of an IgA or an IgM antibody further comprises a CH4domain. The Fc is associated with Fc receptor binding, activation ofcomplement-mediated cytotoxicity and antibody-dependentcellular-cytotoxicity (ADCC). For antibodies such as IgA and IgM, whichare complexes of multiple IgG-like proteins, complex formation requiresFc constant domains.

Finally, the hinge region separates the Fab and Fc portions of theantibody, providing for mobility of Fabs relative to each other andrelative to Fc, as well as including multiple disulfide bonds forcovalent linkage of the two heavy chains.

Antibody “specificity” refers to selective recognition of the antibodyfor a particular epitope of an antigen. The term “epitope” includes anyprotein determinant capable of specific binding to an immunoglobulin orT-cell receptor or otherwise interacting with a molecule. Epitopicdeterminants generally consist of chemically active surface groupings ofmolecules such as amino acids or carbohydrate or sugar side chains andgenerally have specific three dimensional structural characteristics, aswell as specific charge characteristics. An epitope may be “linear” or“conformational”. In a linear epitope, all of the points of interactionbetween the protein and the interacting molecule (such as an antibody)occur linearly along the primary amino acid sequence of the protein. Ina conformational epitope, the points of interaction occur across aminoacid residues on the protein that are separated from one another, i.e.,noncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents, whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen.

Monoclonal antibodies of the present invention may be murine, human,humanized or chimeric. A humanized antibody is a recombinant protein inwhich the CDRs of an antibody from one species; e.g., a rodent, rabbit,dog, goat, horse, or chicken antibody (or any other suitable animalantibody), are transferred from the heavy and light variable chains ofthe rodent antibody into human heavy and light variable domains. Theconstant domains of the antibody molecule are derived from those of ahuman antibody. Methods for making humanized antibodies are well knownin the art. Chimeric antibodies preferably have constant regions derivedsubstantially or exclusively from human antibody constant regions andvariable regions derived substantially or exclusively from the sequenceof the variable region from a mammal other than a human. Thechimerization process can be made more effective by also replacing thevariable regions—other than the hyper-variable regions or thecomplementarity—determining regions (CDRs), of a murine (or othernon-human mammalian) antibody with the corresponding human sequences.The variable regions other than the CDRs are also known as the variableframework regions (FRs). Yet other monoclonal antibodies of the presentinvention are bi-specific, in that they have specificity for both LukAand LukB. Bispecific antibodies are preferably human or humanized.

The above-described antibodies can be obtained in accordance withstandard techniques. For example, LukA, LukB (which as these terms areused herein, include nontoxic analogs thereof such as LukAΔ10C) or animmunologically active fragment of LukA or LukB can be administered to asubject (e.g., a mammal such as a human or mouse). The leukocidins canbe used by themselves as immunogens or they can be attached to a carrierprotein or other objects, such as beads such as sepharose beads. Afterthe mammal has produced antibodies, a mixture of antibody producingcells, such as splenocytes, are isolated, from which polyclonalantibodies may be obtained. Monoclonal antibodies may be produced byisolating individual antibody-producing cells from the mixture andimmortalizing them by, for example, fusing them with tumor cells, suchas myeloma cells. The resulting hybridomas are preserved in culture andthe monoclonal antibodies are harvested from the culture medium.

Techniques for making recombinant monoclonal antibodies are well knownin the art. Recombinant polyclonal antibodies can be produced by methodsanalogous to those described in U.S. Patent Application Publication2002/0009453, using LukA, LukB or LukAB as the immunogen(s).

Therapeutic Compositions

LukA and LukB may be formulated into a therapeutic composition for useas an anti-inflammatory agent in the treatment of acute inflammatoryconditions, including localized acute inflammatory conditions. LukA andLukB may also be formulated into a therapeutic composition for use as anactive vaccine. Anti-LukA and anti-LukB antibodies may be formulatedinto a therapeutic composition for use as a passive vaccine. The passiveand active vaccines may be used prophylactically to inhibit onset of aS. aureus infection, or therapeutically to treat S. aureus infection,particularly S. aureus infections such as MRSA that are known to berefractory or in the case of the specific subject, have provenrefractory to treatment with other conventional antibiotic therapy.

In embodiments wherein the therapeutic composition is intended for useas an active vaccine, the LukA and/or LukB may be altered so as toexhibit reduced toxicity. Molecular alterations are described above.Thus, nontoxic analogs thereof such as LukAΔ10C may be used. Applicantsbelieve that antibodies produced in response to the nontoxic immunogenwill neutralize the toxic, native LukA or LukAB. Other alterations forpurposes of reducing toxicity of LukA and LukB include chemicaltreatment (e.g., modification of specific amino acid residues) orconjugation to another moiety (e.g., to another bacterial antigen, suchas a bacterial polysaccharide or a bacterial glycoprotein). Chemicalalterations to other S. aureus toxins for purposes of inactivation ordetoxification (or reducing toxicity) are known. Methods of determiningwhether a given alteration reduces LukA or LukB toxicity are known inthe art and/or described herein.

The therapeutic compositions of the present invention are prepared byformulating LukA and LukB, or anti-LukA and anti-LukB antibodies, with apharmaceutically acceptable carrier and optionally a pharmaceuticallyacceptable excipient. As used herein, the terms “pharmaceuticallyacceptable carrier” and “pharmaceutically acceptable excipient” (e.g.,additives such as diluents, immunostimulants, adjuvants, antioxidants,preservatives and solubilizing agents) are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Examples of pharmaceutically acceptable carriers include water, e.g.,buffered with phosphate, citrate and another organic acid.Representative examples of pharmaceutically acceptable excipients thatmay be useful in the present invention include antioxidants such asascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; adjuvants (selected so as to avoid adjuvant-inducedtoxicity, such as a β-glucan as described in U.S. Pat. No. 6,355,625, ora granulocyte colony stimulating factor (GCSF)); hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol; saltforming counterions such as sodium; and/or nonionic surfactants such asTWEEN®, polyethylene glycol (PEG), and PLURONICS®.

As described elsewhere herein, the therapeutic compositions of thepresent invention may further contain at least one additional activeagent.

Therapeutic compositions of the present invention may be prepared forstorage by mixing the active ingredient(s) having the desired degree ofpurity with the pharmaceutically acceptable carrier and optionalexcipient and/or additional active agent, in the form of lyophilizedformulations or aqueous solutions.

Uses of the Therapeutic Compositions—Indications

Acute Inflammatory Conditions

Inflammation is generally understood as the protective biologicalresponse to remove harmful invading stimuli such as pathogens (e.g.,bacteria and viruses), damaged cells and irritants, and to initiatehealing. Inflammation is understood more specifically as the reaction ofvascularized living tissue to injury. As such, inflammation is afundamental, stereotyped complex of cytological and chemical reactionsof affected blood vessels and adjacent tissues in response to an injuryor abnormal stimulation caused by a physical, chemical or biologicalagent. Inflammation usually leads to the accumulation of fluid and bloodcells at the site of injury, and is usually a healing process. Withoutthe inflammatory process, wounds and infections would not heal, andprogressive destruction of the tissue would become life-threatening.Acute inflammation refers to the initial response of the body toinvading stimuli and involves the recruitment of plasma and white bloodcells (leukocytes) to the injured or infected tissues. Prolongedinflammation, also referred to as chronic inflammation, involves aprogressive shift in the type of immune cells which are present at thesite of inflammation, and is characterized by simultaneous destructionand healing of the tissue from the inflammatory process.

However, inflammation sometimes causes harm, usually through adysfunction of the normal progress of inflammation. Inflammatorydiseases are those pertaining to, characterized by, causing, resultingfrom, or becoming affected by inflammation. “Acute inflammatoryconditions” as the term is used herein, and in accordance with normalmedical parlance, refers to inflammatory conditions having a rapid onsetand severe symptoms. The duration of the onset, from a normal conditionof the patient to one in which symptoms of inflammation are seriouslymanifested, generally lasts up to about 72 hours. Acute inflammatoryconditions are to be contrasted with chronic inflammatory conditions,which are inflammatory conditions of long duration, denoting a diseaseshowing little change or of slow progression. The distinction betweenacute and chronic conditions is well known to those in the medicalprofessions.

The major immune cells involved in the acute stage of inflammation, aswell as in acute inflammatory disorders, include mononuclear cells(e.g., monocytes, which in response to inflammation differentiate intomacrophages), dendritic cells, and neutrophils (which migrate to theinflammatory site). These immune cells aid in the inflammatory responseby releasing inflammatory mediators such as histamine, interferon-gamma,interleukin-8, leukotriene B4, nitric oxide, etc., and by ingestingbacteria, viruses and cellular debris (a process known as phagocytosis).These cells are known in the art collectively as phagocytes.

Applicants have discovered that LukAB targets and kills human phagocytesand that this LukAB-mediated cytotoxicity is substantially specific tothese cells but not other nucleated mammalian cells. Without intendingto be bound by any particular theory of operation, Applicants believethat the LukA/LukB complex forms pores on the plasma membrane ofinfiltrating phagocytes, causing cell death, thus reducing theinflammation. Thus, the anti-inflammatory compositions of the presentinvention may be useful in treating acute inflammatory conditions inmammals such as humans, regardless of the cause, e.g., any bacterial orviral infection, and in preferred embodiments, localized acuteinflammatory conditions. Other examples of such conditions includeallergic contact dermatitis, acute hypersensitivity, acute neurologicalinflammatory injury (e.g., caused by acute infection), acute myocardialinfarction, acute neuronal injury resulting from cardiopulmonary bypasssurgery, and acute, localized anti-inflammatory conditions caused bybacterial or viral infection.

In preferred embodiments, the acute inflammatory condition is aninfected wound in the skin or soft tissue. Wounds amenable to treatmentwith the invention may be in the form of punctures, cuts or tears of theliving tissues. Wounds of the skin can penetrate the epidermis, dermisor in the case of full-thickness wounds, the subcutaneous tissue. Thus,wounds treatable with the therapeutic compositions of the presentinvention include deep sternal wounds, e.g., following open heartsurgery and post-operative wounds following abdominal and any othertypes of surgery. Other wounds are those which result from trauma suchas by gun shots, knives, or any other object able to cause a cut or tearin the skin. Wounds that arise as a side-effect of medication or as asymptom of various pathologies (e.g., sores associated with Kaposi'sSarcoma), as well as internal wounds (e.g. anal fissures, and wounds orlesions to the gastrointestinal tract, such as ulcers in the stomach orintestines) may also be amenable to treatment with the presentinvention.

Yet other acute inflammatory conditions that may be amenable totreatment with the therapeutic compositions of the present inventioninclude conjunctivitis, iritis, uveitis, central retinitis, externalotitis, acute suppurative otitis media, mastoiditis, labyrinthitis,chronic rhinitis, acute rhinitis, sinusitis, pharyngitis, tonsillitio,contact dermatitis, dermonecrosis, diabetic polyneuritis, polymyositis,myositis ossificans, degenerative arthritis, rheumatoid arthritis,periarthritis scapulohumeralis, and osteitis deformans.

S. aureus Infections

The present invention also provides a method of inhibiting onset of ortreating S. aureus infection by administering the antibody compositionsto a mammalian subject in need thereof. For purposes of the presentinvention, the target subject population includes mammals, such ashumans, who are infected with, or at risk of being infected by S.aureus. In some embodiments, the subject to be treated is infected withS. aureus including MRSA, and/or has already been treated withantibiotics or other therapeutic agents, but the treatment has failed.

Therapeutically Effective Amounts

In the context of treatment of acute inflammatory conditions, theamounts of LukA and LukB are therapeutically effective in the sense thattreatment is capable of achieving any one or more of a reduction in thenumber of symptoms, a decrease in the severity of at least one symptom,or a delay in the further progression of at least one symptom, or even atotal alleviation of the acute inflammatory condition.

In the context of use of the therapeutic compositions as passive oractive vaccines in connection with S. aureus infection, thetherapeutically effective amounts of LukA and LukB, or anti-LukA andanti-LukB antibodies, are also prophylactically effective in the sensethat administration of the composition is capable of achieving any oneor more of inhibition or prevention of an S. aureus infection in thosewho are at risk, and in terms of mammalian subjects infected with S.aureus, a reduction in the number of symptoms, a decrease in theseverity of at least one symptom, or a delay in the further progressionof at least one symptom, or even a total alleviation of the infection.

Broadly, the therapeutically effective amounts of LukA, LukB, andanti-LukA and anti-LukB antibodies can be determined in accordance withstandard procedures, which take numerous factors into account,including, for example, the concentrations of these active agents in thecomposition, the mode and frequency of administration, the severity ofthe acute inflammatory condition or S. aureus infection to be treated(or prevented), and subject details, such as age, weight and overallhealth and immune condition. General guidance can be found, for example,in the publications of the International Conference on Harmonization andin REMINGTON′S PHARMACEUTICAL SCIENCES (Mack Publishing Company 1990). Aclinician may administer LukA and LukB or anti-LukA and anti-LukBantibodies, until a dosage is reached that provides the desired orrequired prophylactic or therapeutic effect. The progress of thistherapy can be easily monitored by conventional assays.

Therapeutically effective amounts of LukA and LukB typically range from1-400 μg of each of LukA and LukB, per dose or on a daily basis.Preferably, the amounts of LukA and LukB are substantially the same.Therapeutically effective amounts of the antibody compositions typicallyare at least 50 mg composition per kilogram of body weight (mg/kg),including at least 100 mg/kg, at least 150 mg/kg, at least 200 mg/kg, atleast 250 mg/kg, at least 500 mg/kg, at least 750 mg/kg and at least1000 mg/kg, per dose or on a daily basis. Dosages for monoclonalantibody compositions might tend to be lower, such as about one-tenth ofnon-monoclonal antibody compositions, such as at least about 5 mg/kg, atleast about 10 mg/kg, at least about 15 mg/kg, at least about 20 mg/kg,or at least about 25 mg/kg.

Modes of Administration

Prior to administration, the therapeutic compositions of the presentinvention may be sterilized which can be readily accomplished byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. Therapeutic compositions may beplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

The anti-inflammatory composition may be administered by any number ofroutes in accordance with accepted medical practice. Preferred modesinclude intravenous, intramuscular, subcutaneous and percutaneousadministration, using techniques that are known in the art. Other routesof administration may be envisioned. In the case of treatment of acuteinflammatory conditions that are localized, non-systemic administrationmay be preferred in which case the administration of the therapeuticcomposition is at or around the site of the acute inflammation.

Combination Therapy

In some embodiments, the therapeutic composition is administered as partof a combination therapy in conjunction with another active agent,depending upon the nature of the acute inflammatory condition or the S.aureus infection that is being treated. Such additional active agentsinclude anti-infective agents, antibiotic agents, and antimicrobialagents. Representative anti-infective agents that may be useful in thepresent invention include vancomycin and lysostaphin. Representativeantibiotic agents and antimicrobial agents that may be useful in thepresent invention include penicillinase-resistant penicillins,cephalosporins and carbapenems, including vancomycin, lysostaphin,penicillin G, ampicillin, oxacillin, nafcillin, cloxacillin,dicloxacillin, cephalothin, cefazolin, cephalexin, cephradine,cefamandole, cefoxitin, imipenem, meropenem, gentamycin, teicoplanin,lincomycin and clindamycin. Dosages of these antibiotics are well knownin the art. See, e.g., MERCK MANUAL OF DIAGNOSIS AND THERAPY, Section13, Ch. 157, 100^(th) Ed. (Beers & Berkow, eds., 2004). Theanti-inflammatory, anti-infective, antibiotic and/or antimicrobialagents may be combined prior to administration, or administeredconcurrently (as part of the same composition or by way of a differentcomposition) or sequentially with the inventive therapeutic compositionsof the present invention.

In some embodiments, the anti-LukA and/or anti-LukB antibody compositionis multivalent in that it also contains an antibody that specificallybinds another bacterial antigen (and that optionally neutralizes theother bacterial antigen). The antibodies may specifically bind any ofthe antigens described herein in the context of the vaccinecompositions. Thus, for example, the other antibody may specificallybind polysaccharides or glycoproteins, including S. aureus Type 5, S.aureus Type 8, S. aureus 336, leukocidin components such as PVL(including the individual PVL subunits, LukS-PV and LukF-PV)gamma-hemolysin subunits (HlgA, HlgB, and HlgC), LukE or LukD from S.aureus, LukM or LukF′-PV from S. aureus, lipoteichoic acid (LTA) andmicrobial surface components recognizing adhesive matrix molecule(MSCRAMM) proteins.

Treatment Regimens

Therapeutic compositions of the present invention may be administered ina single dose, or in accordance with a multi-dosing protocol. Forexample, relatively few doses of the therapeutic composition areadministered, such as one or two doses. In embodiments that includeconventional antibiotic therapy, which generally involves multiple dosesover a period of days or weeks, the antibiotics can be taken one, two orthree or more times daily for a period of time, such as for at least 5days, 10 days or even 14 or more days, while the antibody composition isusually administered only once or twice. However, the different dosages,timing of dosages and relative amounts of the therapeutic compositionand antibiotics can be selected and adjusted by one of ordinary skill inthe art.

Methods of Identifying Inhibitors of LukAB-mediated Cytotoxicity andAltered Forms of LukA and LukB that have Less Toxicity

The anti-LukA and anti-LukB antibodies, and fragments thereof, as wellas other potential therapeutic moieties (e.g., small organic molecules)may be used in various methods (including assay formats or screens) toevaluate their ability to inhibit LukAB-mediated cytotoxicity. Asdescribed below, these methods are designed to identify agents thatinhibit some aspect of the cascade of events that leads toLukAB-mediated cytotoxicity and lysis of human phagocytes. The methodsare also designed to identify altered forms of LukA and LukB thatpossess reduced toxicity relative to their native counterparts. Thetargeted events that are part of the cascade include for example,binding of LukA to phagocyte membranes, binding of LukB to LukA (LukABoligomerization), and blockage of the membrane pore formed by the LukABoligomer. The assay formats generally require human phagocytes (or LukABmembrane-binding portion thereof), suitable culture medium, and purifiedLukA or purified LukA and LukB.

The person of skill will appreciate that the following protocols aremerely illustrative and that various operating parameters such asreaction conditions, choice of detectable label and apparati (e.g.,instrumention for detection and quantification) may be varied as deemedappropriate.

The following methods are generally directed to identifying agents thatinhibit LukAB cytotoxicity, without necessarily revealing the exactevent in the cascade that is affected.

To identify inhibitors of LukAB cytotoxicity, human phagocytes (e.g.,PMN-HL60 cells) may be plated in 384-well clear-bottom black tissueculture treated plate (Corning) at 5×10³ cells/well in a final volume of50 μl of RPMI (Gibco) supplemented with 10% of heat inactivated fetalbovine serum (FBS). Cells may then be contacted/mixed/reacted/treatedwith the test compound/molecule (˜5 μl/different concentrations) andthen intoxicated with LukA and LukB, which in preferred embodiments aresubstantially purified (5 ul of a ˜0.001-2 μM solution), preferablyadded together, under culture conditions to allow for intoxication ofthe phagocytes by LukA and LukB, e.g., for 1 hr at 37° C., 5% CO₂. Ascontrols, cells may be treated with culture medium (100% viable) andwith 0.1% v/v Triton X100 (100% death).

In these embodiments, cells treated as described above may then beincubated with a dye to monitor cell viability (which enablesdetermination of cell viability via absorbance by measuring the numberof viable cells in a culture by quantification of the metabolic activityof the cells) and incubated for an additional time period (e.g., about 2hrs at 37° C., 5% CO₂). Cell viability may then be determined such as bymeasuring the colorimetric reaction at 492 nm using a plate reader e.g.,Envision 2103 Multi-label Reader (Perkin-Elmer). Percent viable cellsmay be calculated such as by using the following equation: %Viability=100×[(Ab₄₉₂Sample−Ab₄₉₂Triton X)/(Ab₄₉₂Tissue culture media).An increase in the 100% viability suggests inhibition of LukABcytotoxicity.

A variation of this assay is referred to as a membrane damage assay. Inthese embodiments, cells treated as described above (e.g., up to andincluding treating of the cells with test compound/molecule and thenintoxicating the cells with purified LukA may then be incubated with acell-impermeable fluorescent dye such as SYTOX green (0.1 μM;Invitrogen) (in accordance with manufacturer's instructions) andincubated e.g., for an additional 15 minutes at room temperature in thedark. Fluorescence, as an indicator of membrane damage, may then bemeasured using a plate reader such as Envision 2103 Multilabel Reader(Perkin-Elmer) at Excitation 485 nm, Emission 535 nm. A decrease influorescence suggests inhibition of LukAB cytotoxicity.

Together these assays facilitate the identification of compounds thatinhibit or reduce LukAB cytotoxic effects towards human phagocyte cells.

Additional methods may be used, independently or in conjunction with themethods described above, particularly if the above methods revealinhibitory activity, that will enable a person skilled in the field todetermine more precisely what event in the biochemical cascade is beingaffected or targeted by the agent. These events include binding of LukAto phagocyte membranes, binding of LukB to LukA (LukAB oligomerization),and blockage of the membrane pore formed by the LukAB oligomer.

Screen for Inhibitors of LukA Binding to Target Cells

To screen for inhibitors that block or reduce LukA binding to targetcells, which is believed to be the first step in the intoxicationprocess, human phagocytes (e.g., PMN-HL60 cells) may be plated in384-well flat-bottom tissue culture treated plates (Corning) at 2.5×10³cells/well in a final volume of 50 μl of RPMI (Gibco) supplemented with10% of heat inactivated fetal bovine serum (FBS). Cells may then betreated with the test compound/molecule (˜5 μl/different concentrations)and intoxicated with purified, fluorescently labeled LukA (e.g., FITC,Cy3, Cy5, APC, PE) 5 ul of a ˜0.01-2 μM solution for 1 hr at 37° C., 5%CO₂. To evaluate the efficacy of the tested compounds/molecules, thecell-associated fluorescence may be measured as an indicator of LukAbinding to cells e.g., using an automated fluorescence microscopicimaging system designed for high content screening and high contentanalysis (e.g., Cellomics ArrayScan HCS Reader (Thermo Scientific)(Excitation 485 nm, Emission 535 nm)).

Screen for Inhibitors of LukA-LukB Oligomerization/Interaction

To screen for inhibitors that block or reduce LukA/LukB interaction,which is believed to be the second step in the intoxication process,human phagocytes (e.g., PMN-HL60 cells) may be plated in 384-wellflat-bottom tissue culture treated plates (Corning) at 2.5×10³cells/well in a final volume of 50 μl of RPMI (Gibco) supplemented with10% of heat inactivated fetal bovine serum (FBS). Cells may then betreated with the test compound/molecule and then intoxicated with amixture of purified LukA and purified LukB where LukB isfluorescently-labeled with a fluorescence molecule such as FITC, Cy3,Cy5, APC, and PE, and allowed to stand to complete the intoxicationprocess (e.g., for 1 hr at 37° C., 5% CO₂). To evaluate the efficacy ofthe tested compounds/molecules, cell-associated LukB-FITC fluorescencemay be measured as an indicator of LukA/LukB-FITC interaction, using forexample, an automated fluorescence microscopic imaging system designedfor high content screening and high content analysis (e.g., a CellomicsArrayScan HCS Reader (Thermo Scientific) (Excitation 485 nm, Emission535 nm)).

Screen for Inhibitors of LukAB Pore Formation

To screen for inhibitors that block or inhibit formation of the LukABpore, the effector molecule that leads to cell lysis, human phagocytes(e.g., PMN-HL60 cells) may be plated in 384-well clear-bottom blacktissue culture treated plate (Corning) at 2.5×10³ cells/well in a finalvolume of 50 μl of RPMI (Gibco) supplemented with 10% of heatinactivated fetal bovine serum (FBS). Cells may then be treated with thetest compound/molecule (˜5 μl containing different concentrations) andthen intoxicated with purified LukAB (˜0.001-2 μM) for 10 minutes at 37°C., 5% CO₂. As controls, PMN-HL60 cells may be treated with culturemedium (negative control) and with 0.1% v/v Triton X100 (positivecontrol).

To directly evaluate LukAB pores on the surface of host cells, anethidium bromide (EB) influx assay may be used. EB is a small-cationicdye that is impermeable into healthy host cells. Upon formation ofcationic pores by LukAB, EB enters the cells and binds to DNA, whichresults in fluorescence. Cell treated in this fashion may then beincubated with EB (5 μM) for an additional 5 minutes at room temperaturein the dark. To evaluate the efficacy of the tested compounds/moleculesin inhibiting LukAB pore-formation the fluorescence may be measured asan indicator of pore-formation, using a plate-reader such as theEnvision 2103 Multilabel Reader (Perkin-Elmer) at Excitation 530 nm,Emission 590 nm. This assay facilitates the identification of moleculesthat can block or inhibit the LukAB pore, which will alleviateLukAB-mediated toxicity.

Method to Determine the Production of LukAB by S. aureus ClinicalIsolates to Predict Severity of Infection

An even further aspect of the present invention is directed to a methodof predicting or assessing severity of an S. aureus infection whichentails detecting presence or amount of LukA and/or LukB, or theircorresponding genes, in a biological sample obtained from an infectedsubject. Thus, detection of presence or relatively high amounts of LukAand/or LukB, or detection of their corresponding genes (e.g., asexhibited by S. aureus strain Newman, 4645, and MRSA strains USA300 andUSA500) relative to a control (e.g., S. aureus strains USA100 andUSA400) which produces little or undetectable amounts of LukA and/orLukB, is indicative of a severe infection. In regard to detection orpresence of relative amounts of LukA and/or LukB, reference may be madeto the illustrations in FIG. 4A. Representative embodiments of themethod are described below.

Immunoblot Analysis to Determine LukA and LukB Levels

To determine LukAB levels (i.e., LukAB production), the biologicalsample e.g., a fluid (e.g., blood) or tissue sample, is obtained fromthe infected subject, followed by exposing the culture to suitableculture conditions to allow for growth of the S. aureus, obtainingculture supernatant, separating bacterial proteins therefrom,identifying LukA and/or LukB, and then quantifying LukA and/or LukB.More specifically, in one embodiment, the clinical isolate strains maybe selected and grown in a suitable culture medium, e.g., Royal ParkMemorial Institute culture medium 1640 (RPMI; Invitrogen) supplementedwith 1% casamino acids (RPMI+CAS) under suitable culture conditions,e.g., for 12-18 hours at 37° C. with shaking at 180 rpm. Bacteria maythen be precipitated via centrifugation and culture supernatantscollected. Culture supernatants (˜30 μl) may then be mixed with 10 μl ofSDS-Laemmli buffer and boiled at 95° C. for 10 minutes. Proteins maythen be separated e.g., using 15% SDS-PAGE gels and then transferred toa solid support, e.g., a nitrocellulose membrane. The membranes may thenbe incubated with antibodies directed against LukA or LukB (e.g., rabbitpolyclonal antibodies), and the presence of LukA or LukB may bevisualized by detecting the antibody-LukA/antibody-LukB complexes with asecondary antibody conjugated to a fluorophore (e.g., anti-rabbitantibody conjugated to AlexaFluor-680; Invitrogen). Membranes may thenbe dried and scanned e.g., using an Odyssey infrared imaging system(LI-COR Biosciences) to determine the amounts of LukA and LukB.Polymerase chain reaction (PCR) to determine the presence of the LukAand/or LukB genes.

To determine the presence of the genes encoding for LukAB, thebiological sample is obtained from the infected subject, followed byexposing the culture to suitable culture conditions to allow for growthof the S. aureus, extracting nucleic acid from the cultured S. aureus,and then conducting at least one round of nucleic acid amplificationusing PCR or other suitable amplification protocol, using LukA and/orLukB-specific primers, and detecting LukA and/or LukB. Thus, in onerepresentative embodiment, following initial sample preparation, theclinical isolate strains may be grown in grown on solid medium e.g.,tryptic soy broth (TSB) solidified with 1.5% agar at 37° C. S. aureuscolonies may then be selected and enzymatically digested, e.g., with 2mg/ml lysostaphin (AMBI PRODUCTS LLC) in TSM buffer (100 mM TRIS pH7,500 mM sucrose, 10 mM MgCl₂)] for 10 minutes at 37° C. Samples may thenbe centrifuged, the supernatant discarded, and the pellet resuspendedwith 100 μl sterile water, followed by boiling for five minutes at 100°C., and centrifugation The supernatant provides the starting materialand the DNA template for an amplification reaction such as PCR usingLukA and/or LukB-specific primers.

WORKING EXAMPLES

The invention will now be described by reference to the followingnon-limiting examples. Unless specified otherwise, all parts are byweight.

Example 1 Expression and Purification of Recombinant LukA and LukB UnderNative Conditions: pMAL Expression System

The LukA and LukB genes were amplified from S. aureus DNA with Taqpolymerase under standard PCR settings with an annealing temperature of55° C. using the following primers:5′-ccc-GTCGAC-tta-TCCTTCTTTATAAGGTTTATTGTC-3′ (SEQ ID NO:30) and5′-ccc-GAAGGATTTCACATCATCATCATCATCACAATTCAGCTCATAAAGACTCTC-3′ (SEQ IDNO:31) for LukA and5′-ccc-CGAAGGATTTCaCATCATCATCATCATCACAAGATTAATTCTGAAATCAAACAAG-3′ (SEQID NO:32) and 5′-ccc-GTCGAC-tta-TTTCTTTTCATTATCATTAAGTACTT-3′ (SEQ IDNO:33) for LukB. The LukA and LukB gene products were digested with Nde1and Sal1 (New England BioLabs) and ligated into the pMAL-c4X vector (NewEngland BioLabs). The constructs were transformed into the E. colistrain DH5α and the plasmid inserts were confirmed through sequencing.The transformants were grown in Terrific Broth with 100 ug/ml ofampicillin and 0.2% glucose at 37° C. until cultures reached an A₆₀₀ of˜0.5. The expression of 6-his-tagged MBP-LukA or 6-his-tagged MBP-LukBwas induced with 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at16° C., overnight, with 180 rpm shaking.

After the induction, the cells were harvested through centrifugation at4000 rpm at 4° C. for 20 min and resuspended in ice cold Column Buffer(20 mM Tris-HCL, 200 mM NaCl, and 1 mM EDTA) supplemented with EDTA-freeprotease inhibitor (Roche). The bacterial cells were sonicated on icefor 1 min (10 sec pulses). The samples were centrifuged at 10,000 rpm at4° C. for 30 min and the supernantant was collected and applied to anamylose resin column. The columns were washed two times with ColumnBuffer and purified 6-his-tagged MBP-LukA or 6-his-tagged MBP-LukB waseluted in 10 fractions with Column Buffer supplemented with 10 mMmaltose.

Example 2 Expression and Purification of Recombinant Active LukA,LukAΔ10C, Δ33NLukA and LukB Toxins: pET14b Expression System

The LukA, LukAΔ10C, Δ33NLukA and LukB genes were amplified from S.aureus DNA with Vent polymerase (New England BioLabs) under standard PCRsettings with an annealing temperature of 55° C. using the followingprimers: 5′-cccc-CTCGAG-AATTCAGCTCATAAAGACTCTCAAG-3′ (SEQ ID NO:34) and5′-cccc-GGATCC-tta-TCCTTCTTTATAAGGTTTATTGTC-3′ (SEQ ID NO:35) for LukA;5′-cccc-CTCGAG-AATTCAGCTCATAAAGACTCTCAAG (SEQ ID NO:34) and5′-cccc-GGATCC-tta-ATATTTATCAACGACTTTAACTG (SEQ ID NO:36) for LukAΔ10C;5′-cccc-CTCGAG-TCAACAGCACCGGATGATATTG (SEQ ID NO:37) and5′-cccc-GGATCC-tta-TCCTTCTTTATAAGGTTTATTGTC (SEQ ID NO:35) for Δ33NLukA;5′-cccc-CTCGAG-AAGATTAATTCTGAAATCAAACAAG-3′ (SEQ ID NO:38) and5′-cccc-GGATCC-tta-TTTCTTTTCATTATCATTAAGTACTTT-3′ (SEQ ID NO:39) forLukB. The gene products were digested with Xho1 and BamH1 (New EnglandBioLabs) and ligated into the pET14b vector (Novagen) fusing the codingsequence of a histidine-tag to the 5′-region of the genes. Theexpression plasmids were transformed into the E. coli strain DH5α andthe plasmid inserts were confirmed through sequencing. The plasmids werepurified and transformed into the expression E. coli strain T7 lysY/lq(New England BioLabs).

For purification under denaturing conditions the transformants weregrown in Terrific Broth with 100 μg/ml of ampicillin at 37° C. untilcultures reached an A600 of ˜0.5. The expression of 6-his-tagged LukA or6-hisLukB was induced with 0.4 mM IPTG at 37° C., for 3 hrs, with 180rpm shaking. After the induction, the cells were harvested throughcentrifugation at 4000 rpm at 4° C. for 15 min and then resuspended in1×TBS (50 mM Tris, 150 mM NaCl, pH 7.5). The bacterial cells weresonicated on ice for 2 min (10 sec pulses). The sonicated bacteria wereultracentrifuged for 30 min at 50,000 rpm. The pellets were resuspendedin lysis buffer (100 mM NaH2PO4, 10 mM Tris, 8M urea, pH 8.0) andincubated at room temperature for 30 min on a rotisserie. The sampleswere centrifuged at 13,000 rpm for 30 min and the supernatants wereapplied to a column containing Ni-NTA resin (Qiagen). The column waswashed two times with wash buffer (100 mM NaH₂PO₄, 10 mM Tris, 8M urea,pH 6.3) and the protein was eluted from the column using elution buffer(100 mM NaH₂PO₄, 10 mM Tris, 8M urea) at pH 5.9 and at pH 4.5. Thefractions containing purified protein, as determined by SDSPAGE, werepooled, diluted 1:1 in tris buffered saline (TBS; 500 mM Tris, 1.5MNaCl, pH 7.5), and dialyzed in TBS at 4° C. overnight to remove the ureaand allow refolding. Purified 6-his-tagged LukA and LukB were quantifiedusing the Thermo Scientific Pierce BCA Protein Assay Kit.

For purification under native conditions the transformants were grown inLuria-Bertani broth with 100 μg/ml of ampicillin at 37° C. untilcultures reached an A600 of ˜0.5. The expression of 6-his-tagged LukA,6-his-tagged LukAΔ10C, 6-his-tagged Δ33NLukA or 6-hisLukB was inducedwith 0.05-0.1 mM IPTG at 25-30° C., for 3 hrs, with 220 rpm shaking.After the induction, the cells were harvested through centrifugation at4000 rpm at 4° C. for 15 min and then resuspended in 1×TBS (50 mM Tris,600 mM NaCl, pH 7.5) with 10 mM imidazole and HALT EDTA-free proteaseinhibitor cocktail (Thermo Scientific). The bacterial cells weresonicated on ice. The sonicated bacteria were centrifuged for 20 min at20,000 rpm. The supernatants were incubated with Ni-NTA resin (Qiagen)for 1 hr at 4° C. on a rotisserie. The samples were applied to a columnand the column was washed with wash buffer 1×TES (50 mM Tris, 600 mMNaCl, pH 7.5) with 25 mM imidazole. The protein was eluted from thecolumn using 50-500 mM imidazole in elution buffer 1×TBS (50 mM Tris,600 mM NaCl, pH 7.5). The fractions containing purified protein, asdetermined by SDS-PAGE, were pooled, diluted 1:1 in 1×TBS (50 mM Tris,150 mM NaCl, pH 7.5), and dialyzed in 1×TBS at 4° C. overnight. Purified6-his-tagged LukA and LukB were quantified using the Thermo ScientificPierce BCA Protein Assay Kit.

Example 3 Expression and Purification of Denatured Recombinant LukA andLukB

The LukA and LukB genes were amplified from S. aureus DNA with Ventpolymerase (New England BioLabs) under standard PCR settings with anannealing temperature of 55° C. using the following primers:5′-ggg-CATATG-AATTCAGCTCATAAAGACTCTCAA-3′ (SEQ ID NO:40) and5′-ccc-GTCGAC-TCCTTCTTTATAAGGTTTATTGTC-3′ (SEQ ID NO:41) for LukA and5′-ggg-CATATG-AAGATTAATTCTGAAATCAAACAAG-3′ (SEQ ID NO:42) and5′-ccc-GTCGAC-TTTCTTTTCATTATCATTAAGTACTT-3′ (SEQ ID NO:43) for LukB. TheLukA and LukB gene products were digested with Nde1 and Sal1 (NewEngland BioLabs) and ligated into the pET41b vector (Novagen). Theconstructs were first transformed into DH5α cells and then transformedinto the E. coli expression strain ER2566 (New England BioLabs). Thetransformants were grown in Terrific Broth with kanamycin, 25 ug/ml, for2.5 hrs at 37° C. and expression of LukA and LukB was induced with 0.3mM IPTG at 37° C. for 2 hrs with 180 rpm shaking. The cells werepelleted and resuspended in 1×TBS (500 mM Tris, 1.5M NaCl, pH 7.5) andsonicated on ice for 1 min (10 sec pulses). The sonified bacteria wereultra-centrifuged for 30 min at 50,000 rpm.

In order to purify the C-terminal 6-his-tagged LukA and LukB underdenaturing conditions, the pellets were resuspended in lysis buffer (100mM NaH₂PO₄, 10 mM Tris, 8M urea, pH 8.0) and incubated at roomtemperature for 30 min on a rotisserie. The samples were centrifuged at13,000 rpm for 30 min and the supernantants were applied to a Ni-NTAcolumn. The columns were washed two times with wash buffer (100 mMNaH₂PO₄, 10 mM Tris, 8M urea, pH 6.3) and LukA and LukB were eluted fromthe columns using elution buffer (100 mM NaH₂PO₄, 10 mM Tris, 8M urea)at pH 5.9 and at pH 4.5. Purified 6-his-tagged LukA and LukB werequantified using the BioRad DC Protein Assay.

Example 4 Production of Anti-LukA and Anti-LukB Polyclonal Antibodies

Denatured-recombinant LukA (250 μg) emulsified in Freund's CompleteAdjuvant (FCA) was injected into New Zealand White rabbits. Animals wereboosted with recombinant LukA (125 μg) emulsified in Incomplete Freund'sAdjuvant (FCA) at day twenty one (21) and at day forty-nine (49).

Denatured-recombinant LukB (250 μg) emulsified in Freund's CompleteAdjuvant (FCA) was injected into New Zealand White rabbits. Animals wereboosted with recombinant LukB (125 μg) emulsified in Incomplete Freund'sAdjuvant (FCA) at day twenty one (21) and at day forty-nine (49).

Example 5 Leukocidin A/B is Predominantly Responsible for theCytotoxin-mediated Killing of Human Phagocytes Through MembraneDisruption Cell Lines Used

As a model to study how LukAB targets and kills human phagocytes HL-60cells (ATCC CCL-240, a human promyelocytic cell line), were used. HL-60cells were grown in RPMI medium (Gibco) supplemented with 10% ofheat-inactivated fetal bovine serum (FBS) at 37° C. with 5% CO₂. Todifferentiate HL-60 into neutrophil-like cells (PMN-HL60), cultures weresupplemented with 1.5% (v/v) dimethylsulfoxide (DMSO) and grown for 4days.

Methods/Assays Used

Cell Toxicity Assay

To evaluate the viability of mammalian cells after intoxication withStaphylococcus aureus leukocidin AB (LukAB), PMN-HL60 cells were platedin 96-well flat-bottom tissue culture treated plates (Corning) at 1×10⁵cells/well in a final volume of 100 ul of RPMI (Gibco) supplemented with10% of heat inactivated fetal bovine serum (FBS). Cells were intoxicatedfor 2 hrs at 37° C., 5% CO₂ with serial 2-fold dilutions of culturefiltrate from Staphylococcus aureus strain Newman ranging from 20% to0.16% v/v in triplicate. Experiments were performed using exoproteinsfrom a wild-type strain and exoproteins from a strain lacking LukAB(mutant strain). Controls for 100% viability included 20% v/v tissueculture media (RPMI+10% heat-inactivated fetal bovine serum), and 20%v/v S. aureus growth media (RPMI+Cas amino acids). 0.1% v/v Triton X100was used as a control for 100% cell death. After the intoxication, 10 μlof a dye suitable for monitoring cell viability was added to each welland the cells were incubated for an additional 3 hrs at 37° C., 5% CO₂.This dye monitors metabolically active cells (color change), a propertylost in dead cells. The colorimetric reaction was measured at 492 nmusing a Perkin Elmer Envision 2103 Multilabel Reader. Percent viablecells were calculated using the following equation: %Viability=100×[(Ab₄₉₂Sample−Ab₄₉₂Triton X)/(Ab₄₉₂Tissue culture media).

Membrane Damage Assay

An alternative assay to measure LukAB-mediated cytotoxicity is toevaluate the integrity of host cell membranes. To this end, a SYTOXgreen (Invitrogen) permeability assay was employed. Healthy cells areimpermeable to SYTOX green, but become permeable to the dye once thecell membrane integrity has been compromised. Inside the cells, SYTOXgreen binds to DNA and exhibits strong fluorescence.

To evaluate the integrity of host cell membranes after intoxication withLukAB or ex vivo infection with S. aureus strains, PMN-HL60 cells wereplated in 96-well flat-bottom tissue culture treated plates (Corning) at1×10^5 cells/well in a final volume of 100 ul of RPMI (Gibco)supplemented with 10% of heat inactivated fetal bovine serum (FBS).Cells were either intoxicated with dilutions of culture filtrate from S.aureus strain Newman ranging from 20% to 0.16% v/v or infected with MOIsranging from 1-100 in triplicate for 2 hrs at 37° C., 5% CO₂.Experiments were performed using a wild-type strain and a strain lackingLukAB (mutant strain). Controls for background fluorescence included 20%v/v tissue culture media (RPMI+10% heat-inactivated fetal bovine serum)and 20% v/v S. aureus growth media (RPMI+Cas Amino acids). After theintoxication or infection, the cells were transferred to 96-wellv-bottom plate (Corning) and centrifuged at 1500 rpm for 5 min. Thesupernatant was discarded and the pellets were resuspended in 100 ul ofPBS+SYTOX green (0.1 μM; Invitrogen). The cells were then transferred to96-well clear-bottom black tissue culture treated plate (Corning) andincubated at room temperature in the dark for 10 min. Fluorescence wasmeasured using a Perkin Elmer Envision 2103 Multilabel Reader(Excitation 485 nm, Emission 535 nm).

Results

LukAB Targets and Kills Human Primary Phagocytes

Intoxication of primary human peripheral mononuclear cells (PBMCs) (FIG.3a ), monocytes, macrophages, dendritic cells (FIG. 3b ) andpolymorphonuclear cells (PMNs) (FIG. 3c ) with filtered culturesupernatants from the S. aureus strain Newman resulted in potent celldeath as examined by the Cell Toxicity Assay (FIGS. 3a-c ). Intoxicationof these cells with filtered culture supernatants from the S. aureusstrain Newman lacking-hemolysin (hla), γ-hemolysin (hlg), leukocidin E/D(LukED) or leukocidin A/B (LukAB) revealed that exoproteins from hla-,hlg-, and LukED-negative strains are as cytotoxic as the Newman wildtype(WT) strain (as examined by the cell toxicity assay), indicating none ofthe previously-described leukotoxins produced by Newman contributes tothe cytotoxin-mediated killing of these cells (FIGS. 3a-c ). Incontrast, very little cell death was observed when cells wereintoxicated with exoproteins from the strain Newman lacking LukAB(ΔlukAB). The lack of cytotoxic activity by the strain Newman lackingLukAB was rescued by providing the lukAB genes in trans in a plasmid(ΔlukAB/pLukAB) (FIG. 3c ). Importantly, this phenotype is fullydependent on LukAB as determined by intoxicating PMNs withpurified-recombinant LukA and LukB. Individual subunits exhibited nodetectable cytotoxicity towards PMNs (FIG. 3d ). In contrast,combination of both subunits resulted in potent cytotoxicity towardsthese cells in a dose-dependent manner (FIG. 3d ). Altogether, theseresults demonstrate LukAB is responsible for the ability of S. aureus totarget and kill primary human phagocytes, key immune cells required forprotecting the host against infectious agents.

LukAB Preferentially Kills Human Phagocytic Cells

Intoxication of the neutrophil-like cell line (PMN-HL60) and themacrophage-like cell line (THP1+PMA) with filtered culture supernatantsfrom the S. aureus strain Newman resulted in potent cell death asexamined by the Cell Toxicity Assay (FIG. 3). Intoxication of thesecells with filtered culture supernatants from the S. aureus WT and theisogenic cytotoxin mutant strains (hla, hlgABC, LukED, and LukAB)revealed that that LukAB is responsible for the ability of S. aureus tokill these cells as determined by the cell toxicity assay (FIGS. 4a and4b ). The lack of cytotoxic activity by the strain Newman lacking LukABwas rescued by transforming the strain Newman lacking LukAB with aplasmid expressing lukAB (ΔlukAB/pLukAB) (FIG. 4c ). Exoproteins fromthis strain were extremely cytotoxic to both PMN-HL60 cells andTHP-1+PMA cells, providing strong evidence that LukAB is a potentstaphylococcal toxin that targets and kills human cells.

To further rule out the contribution of other factors present in S.aureus culture supernatant, PMN-HL60 cells were intoxicated withpurified-recombinant LukA or LukB. Individual subunits exhibited nodetectable cytotoxicity towards PMN-HL60 (FIG. 3d ). In contrast,combination of both subunits resulted in potent cytotoxicity towardsthese cells in a dose-dependent manner (FIG. 3d ). In addition toPMN-HL60s and THP-1+PMA cells, several other human cell lines includingthe myeloid progenitor that PMN-HL60s are differentiated from (HL60),the monocyte progenitor that THP-1+PMA are differentiated from (THP-1),lymphocytes (HuT and Jurkat cells), and epithelial cells (293T andHepG2) were also intoxicated with recombinant LukAB (FIG. 4e ). Theseresults demonstrate that LukAB preferentially targets and kills humanphagocytic cells and has no effect on human lymphocytes or epithelialcells. Together these results demonstrate that LukAB plays a significantrole in S. aureus-mediated killing of phagocytes.

LukAB is Produced by Clinical Relevant Strains of S. aureus

Immunoblot analyses with a polyclonal antibody raised against LukBrevealed that LukB is produced by a series of staphylococcal strainsincluding MRSA strains associated with hospital- and community-acquiredinfections (USA300, 400, and 500; FIG. 5a ). Importantly, LukB levelsare associated with the cytotoxic phenotype of these strains (FIGS. 5aand 5b ). Strains that produce high levels of LukB (e.g., Newman, 4645,USA 500, and USA 300) were more cytotoxic towards PMN-HL60 cells thanstrains that produce low or undetectable LukB (e.g., USA100 and USA400)(FIG. 5b ). To investigate the role of LukAB in MRSA strains, a LukABisogenic mutant was created in the clinical isolate USA type 300 LAclone (FIG. 5c ). As seen with strain Newman, exoproteins from strainUSA300 lacking LukAB were noticeably less cytotoxic than exoproteinsfrom the parental strain (FIG. 5d ). These data demonstrate that LukABis an important cytotoxin produced by MRSA strains.

LukAB Damages the Membranes of Human Phagocytes

Intoxication of PMN-HL60 with exoproteins from S. aureus resulted incell rounding and nuclear swelling, a phenotype dependent on LukAB (FIG.6a ). This cell rounding and swelling phenotype was associated withincreased membrane permeability as determined by the Membrane DamageAssay (FIGS. 6b and 6c ). Importantly, exoproteins from theLukAB-negative strain exhibited little to no effect on membranepermeability, a phenotype that was rescued by producing LukAB from aplasmid (FIG. 6b ) and recombinant LukAB but the not the individualtoxin subunits cause membrane damage in a dose-dependent manner (FIG. 6c). Furthermore, infection of primary human PMNs with both amethicillin-sensitive S. aureus (MSSA) strain and amethicillin-resistant S. aureus (MRSA) USA300 strain resulted inLukAB-dependent membrane damage (FIG. 6d ). These results demonstratethat LukAB damages the plasma membrane of host cells during ex vivoinfection.

LukAB protects S. aureus from host-mediated killing, by targeting andkilling phagocytes.

Infection of PMN-HL60 cells with S. aureus WT, ΔlukAB and the ΔlukABharboring the lukAB expression plasmid (ΔlukAB/pLukAB) revealed thatLukAB is required for the ability of S. aureus to disrupt the membraneof phagocytes during staph-phagocyte interaction (FIG. 7a ), asdetermined by the Membrane Damage Assay. Importantly, S. aureusoverproducing LukAB (ΔlukAB/plukAB) exhibited more membrane damage thanthe WT strain (FIG. 7a ) demonstrating that LukAB potently damages hostcell membranes. Infection of human whole blood (FIG. 7b ) and purifiedprimary human neutrophils (PMN; FIG. 7c ) revealed that lukAB-negativestaph was killed more efficiently compared to the WT strain (FIGS. 7band 7c ). Importantly, the attenuated phenotype exhibited by theΔlukAB-negative staph was rescued with the lukAB expression plasmid(FIGS. 7b and 7c ). Intoxication of primary human PMNs with culturefiltrate of S. aureus WT, ΔlukAB, and the ΔlukAB mutant straincontaining the lukAB expression plasmid revealed that LukAB targets andkills primary human PMNs (FIG. 7d ). These data strongly indicate thatLukAB is a potent staphylococcal cytotoxin that targets and kills PMNsthrough membrane disruption thus protecting S. aureus from PMN-mediatedkilling.

LukAB Contributes to the Pathogenesis of S. aureus In Vivo

Mice infected retro-orbitally with S. aureus containing a luciferasereporter construct with the LukAB promoter fused to it (pLukAB-Xen1)showed LukAB promoter activity in kidney abscesses, where as apromoterless reporter (pXen1) showed no activity (FIG. 8a ). These datademonstrate that LukAB is expressed in vivo in a renal abscess model ofinfection. In addition, mice infected retro-orbitally with S. aureus WTbut not a S. aureus strain lacking LukAB displayed extensivecolonization of the kidneys. The colonization defect observed in theLukAB-negative strain was restored to WT levels by providing LukAB intrans (FIG. 8b ). Together these data show that LukAB is an importantstaphylococcal cytotoxin that contributes to the pathogenesis of thebacterium.

LukAB Forms Pores on the Target Cell Membrane that can be Blocked withPolyethylene Glycol

Intoxication of PMN-HL60 cells with recombinant LukAB (rLukAB) revealedthat both rLukA and rLukB bind to target cells as determined viaimmunoblot with LukA and LukB specific antibodies (FIG. 9a ). Binding of6His-tagged rLukAB to PMN-HL60s was also confirmed usingfluorescence-activated cell sorting (FACS) and a His specific antibody(FIG. 9b ). rLukAB oligomers were also detected via immunoblot onPMN-HL60 membranes after intoxication with the recombinant toxinindicating that LukAB forms higher order structures on target-cellmembranes (FIG. 9c ). Importantly, intoxication of PMN-HL60s with rLukABdemonstrated that LukAB forms ethidium bromide permeable pores ontarget-cell membranes that can be blocked using polyethylene glycolmolecules (PEG) (FIG. 9d ), and that blocking the LukAB pores increasesviability of the cells (FIG. 9e ). Furthermore, the PEGs specificallyblock LukAB pores, as pores formed in PMN-HL60 membranes by saponin werenot blocked by the PEGs and as a result these cells were not protectedfrom pore-mediated death. These data demonstrate that LukAB pores can beblocked by small molecules and blocking LukAB pores protects cells fromLukAB-mediated killing.

Neutralization of S. aureus Culture Filtrate Cytotoxicity with an α-LukAPolyclonal Antibody.

Intoxication of PMN-HL60s with S. aureus culture filtrate pre-incubatedwith various amounts of α-LukA polyclonal antibodies generated in twodifferent rabbits resulted in decreased toxicity of the culture filtratein a dose-dependent manner (FIG. 10). This neutralizing effect was notseen when culture filtrate was pre-incubated with pre-immune serum.Importantly, antibodies generated in the two different rabbits behavedvery similar, and the neutralization capabilities of the antibodiesincreased with maturity as seen by comparing the neutralizing effect ofantibody from the late bleeds to the neutralizing effect of antibodyfrom the early bleeds (FIG. 10). These data show that cytotoxicity seenwith culture filtrate from S. aureus can be neutralized with α-LukApolyclonal antibodies.

Identification of a Non-cytotoxic LukA Truncation Mutant that is StillRecognized by the α-LukA Polyclonal Antibody.

LukA differs from the other staphylococcal leukotoxin S-subunits in thatit has an extension at both the N- and C-terminus. This extensionconsists of 33 amino acids at the N-terminus and 10 amino acids at theC-terminus (FIG. 11a ). Intoxication of PMN-HL60s with purifiedrecombinant LukA lacking the N-terminus extension (rΔ33NLukA) incombination with purified rLukB resulted in potent cytotoxicity towardsthe cells comparable to that of purified rLukA+rLukB (FIG. 11b ).However, intoxication of PMN-HL60s with purified recombinant LukAlacking the C-terminal extension (rLukAΔ10C) in combination with rLukBresulted in no cytotoxic effect (FIG. 11b ). These data demonstrate thatthe N-terminal extension is dispensable for the cytotoxic effect of LukAbut the C-terminal extension is necessary for toxicity. Importantly, theα-LukA polyclonal antibody that neutralizes the effect of LukAB (FIG.10) still recognizes the 6×His-tagged non-cytotoxic rLukAΔ10C mutantjust as well as the α-His polyclonal antibody (FIG. 11c ). These datasuggest that rLukAΔ10C may be exploited to generate α-LukA polyclonalantibodies in vivo that are neutralizing antibodies. Therefore,rLukAΔ10C may be used in an active vaccine composition.

All patent publications and non-patent publications are indicative ofthe level of skill of those skilled in the art to which this inventionpertains. All these publications are herein incorporated by reference tothe same extent as if each individual publication were specifically andindividually indicated as being incorporated by reference.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

The invention claimed is:
 1. A composition comprising: a LukApolypeptide analog, wherein said analog comprises the amino acidsequence of amino acid residues 28-341 of SEQ ID NO: 2, and does notcomprise the amino acid sequence of amino acid residues 342-351 of SEQID NO: 2, wherein said LukA polypeptide analog is non-cytotoxic whencombined with a LukB polypeptide comprising the amino acid sequence ofamino acid residues 30-339 of SEQ ID NO:
 27. 2. The composition of claim1 further comprising a LukB polypeptide comprising the amino acidsequence of amino acid residues 30-339 of SEQ ID NO:
 27. 3. Thecomposition of claim 1 further comprising a pharmaceutically acceptablecarrier.